A-deoxy-a-aza derivatives of clavulanic acid, a process for their preparation and their use

ABSTRACT

The compounds of the formula (II): ##STR1## or pharmaceutically acceptable salts or esters thereof wherein A is a hydrogen atom or an esterifying radical; X is an alkyl group of 1-12 carbon atoms optionally substituted by a hydroxy, amino, acylamino of C 1-6  alkoxy group, which substituents are not on the carbon atom adjacent to the nitrogen atom; or a C 5-7  cycloalkyl group; or a phenylalkyl group wherein the carbon atom content of the alkyl part is 1-6 and the phenyl part is optionally substituted with a fluorine, bromine, chlorine, C 1-6  alkyl, or C 1-6  alkoxy; with the proviso that when X represents an optionally substituted phenylalkyl group and A represents C 1-3  alkyl, then the --CO 2  A group is attached to the alkyl part of the phenylalkyl group; have been found to be β-lactamase inhibitors and antibacterial agents. Their preparation and use is described.

CROSS-REFERENCE

This is a continuation of Ser. No. 176,161 filed Aug. 7, 1980, nowabandoned.

This invention relates to a novel class of clavulanic acid derivativesand in particular to a class of carboxyl-substituted alkylaminederivatives and carboxyl-substituted aralkylamine derivatives ofclavulanic acid. These compounds have antibacterial and β-lactamaseinhibitory properties and are therefore of use in the treatment ofbacterial infections either alone or in combination with otherantibacterial agents such as penicillins or cephalosporins.

Some carboxyl-substitued aralkylamine derivatives are known from WGerman OLS No. 2817085 (equivalent to French Pat. No. 2387986, JapanesePatent Application No. 48292/78 and U.S. Patent Application Ser. No.896,441) and European Application No. 79301618.9. W German OLS No.2817085 discloses inter alia compounds of formula (I): ##STR2## whereinR³ and R⁴ represent inter alia hydrogen, fluorine, chlorine, C₁₋₃ alkylor C₁₋₃ alkoxy, and R² represents inter alia an alkoxycarbonyl groupcontaining 1-3 carbon atoms in the alkoxy part.

European Application No. 79301618.9 discloses inter alia compounds offormula (IA): ##STR3## wherein R², R³ and R⁴ are as defined above.

The present invention provides the compounds of the formula (II):##STR4## or a pharmaceutically acceptable salt or ester thereof whereinA is a hydrogen atom or an esterifying radical; X is an alkylene groupof 1-12 carbon atoms optionally substituted by a hydroxy, amino,acylamino or C₁₋₆ alkoxy group, which substituents are not on the carbonatom adjacent to the nitrogen atom; or a C₅₋₇ cycloalkylene group; or aphenylalkylene group wherein the carbon atom content of the alkylenepart is 1-6 and the phenyl part is optionally substituted with afluorine, bromine, chlorine, C₁₋₆ alkyl, or C₁₋₆ alkoxy; with theproviso that when X represents an optionally substituted phenylalkylenegroup and A represents C₁₋₃ alkyl, then the --CO₂ A group is attached tothe alkyl part of the phenylalkyl group.

Suitably X is a C₁₋₁₂ alkylene group optionally substituted with ahydroxy, amino, acylamino or C₁₋₆ alkoxy group which substituents arenot on the carbon atom adjacent to the nitrogen atom; or a C₅₋₇cycloalkylene group.

The group X may be an optionally substituted alkylene group such asmethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, decylene, hydroxyethylene, hydroxypropylene, hydroxybutylene,methoxyethylene, methoxypropylene, ethoxyethylene, propoxyethylene,aminopentylene. A preferred cycloalkyl group is cyclohexyl. Preferably Xis C₁₋₆ alkylene.

When the group X is optionally substituted phenylalkylene, the group--CO₂ A may be attached either to the phenyl part or the alkylene part.Suitable such groups include benzyl, phenylethylene, phenylpropylene,phenylbutylene, fluorobenzyl, chlorobenzyl, bromobenzyl, methylbenzyl,ethylbenzyl, methoxybenzyl, ethoxybenzyl or hydroxybenzyl.

Alternatively X may represent a benzyl, bromobenzyl, chlorobenzyl,fluorobenzyl, methylbenzyl, methoxybenzyl or hydroxybenzyl group.

More suitably X is a phenylethylene, phenylpropylene or phenylbutylenegroup.

Preferred groups X include the ethylene, propylene, butylene and benzylgroups.

A particularly preferred group X is ethylene.

A further particularly preferred group X is benzyl.

It is realised that there are two carboxylate groups in the compounds ofthe formula (II), thus the compounds of this invention may be presentedin the form of a di-ester. Alternatively the compounds of the formula(II) may have one carboxylate function esterified and the othersalified. In a further form the compounds of the formula (II) may bepresented as di-salts. It is to be realised also that reference to asalt hereinabove covers the compounds of the formula (II) when inzwitterionic form.

In a preferred aspect of this invention the compounds of the formula(II) are presented in the form of a zwitterion wherein A is a hydrogenatom or an esterifying radical.

In a further preferred aspect of this invention the compounds of theformula (II) are presented in the form of a mono-ester wherein A is ahydrogen atom or a salting group.

In a further preferred aspect of this invention the compounds of theformula (II) are presented in the form of a di-salt.

Suitable pharmaceutically acceptable salting groups that may be presentin a compound of this invention include the sodium, potassium andcalcium ions.

Preferably the pharmaceutically acceptable salting group present in acompound of this invention is either a sodium or potassium ion.

Certain suitable derivatives of the compounds of the formula (II)include those of the formula (III): ##STR5## wherein A is a hydrogenatom, or an alkyl group of 1-6 carbon atoms optionally substituted by analkoxy or acyloxy group of 1-7 carbon atoms, or is a group of thesub-formula (b):

    CHA.sub.2 A.sub.3                                          (b)

wherein A₂ is an alkenyl or alkynyl group of up to 5 carbon atoms or isa phenyl group optionally substituted by a fluorine, chlorine, bromine,nitro or alkyl or alkoxyl of up to 4 carbon atoms; and A₃ is a hydrogenatom, an alkyl group of up to 4 carbon atoms or a phenyl groupoptionally substituted by a fluorine, chlorine, bromine, nitro or alkylor alkoxyl of up to 4 carbon atoms; and A₁ is as defined for Ahereinabove.

Suitable values for A or A₁ include a hydrogen atom and the methyl,ethyl, n-propyl, n-butyl, allyl, CH₂ --C═CH, methoxymethyl,acetoxymethyl, propionoxymethyl,pivaloyloxymethyl,ethoxycarbonyloxymethyl, methoxycarbonyloxyethyl,ethoxycarbonyloxyethyl, dimethoxyphthalidyl, benzyl, methoxybenzyl,ethoxybenzyl, nitrobenzyl and chlorobenzyl groups.

Certain favoured values A or A₁ include a hydrogen atom and the methyl,ethyl, propyl, methoxy; methyl, acetoxymethyl, acetoxyethyl, phthalidyl,ethoxycarbonyloxymethyl, and α-ethoxycarbonyloxyethyl groups.

Certain favoured groups A₂ include the phenyl and 4-methoxyphenylgroups. A particularly favoured moiety A₃ is the hydrogen atom.

Certain other favoured values for A or A₁ include those of thesub-formulae (c), (d) and (e):

    --CHA.sub.5 --OA.sub.6                                     (c)

    --CHA.sub.5 --COA.sub.6                                    (d)

    --CHA.sub.5 --CO.sub.2 A.sub.6                             (e)

wherein A₅ is a hydrogen atom or a methyl grop and A₆ is an alkyl groupof up to 4 carbon atoms or a phenyl or benzyl group either of which maybe substituted by one or two alkyl or alkoxyl groups of up to 3 carbonatoms or by a fluorine, chlorine or bromine atom or a nitro group; or A₅is joined to A₆ to form the residue of an unsubstituted saturated 5- or6-membered heteroalicyclic ring or an orthophenylene group which may besubstituted by one or two alkyl or alkoxyl groups of up to 3 carbonatoms or by a fluorine, chlorine or bromine atom or nitro group.

An apt acyclic value for the sub-group of the formula (c) is --CH₂--OA₆.

An apt acyclic value for the sub-group of the formula (d) is --CH₂--CO--A₆.

An apt acyclic value for the sub-group of the formula (e) is --CH₂ --CO₂A₆.

A further apt acyclic value for the sub-group of the formula (e) is--CH(CH₃)--CO₂ A₆.

Favoured values for A₆ in the preceding acyclic moieties include themethyl, ethyl, propyl, butyl, phenyl and benzyl groups.

Apt cyclic values for the sub-group of the formula (d) include thetetrahydropyranyl and tetrahydrofuranyl groups.

Di-esters of the compounds of the formula (II) may be presented in theform of their acid addition salts if desired. The acid used to form thesalt will most suitably be pharmaceutically acceptable, butnon-pharmaceutically acceptable acid addition salts are also envisaged,for example as intermediates in the preparation of the pharmaceuticallyacceptable salts by ion exchange. Suitable pharmaceutically acceptableacid addition salts include those of inorganic and organic acids, suchas hydrochloric, phosphoric, sulphuric, methanesulphonic,toluenesulphonic, citric, malic, acetic, lactic, tartaric, propionic,succinic or the like acid. Most suitably the acid addition salt isprovided as a solid and preferably as a crystalline solid.

An especially suitable form of the compounds of the formula (III) isthat in which A is a hydrogen atom or an esterifying radical and A₁ is ahydrogen atom.

Compounds when in this form tend to exist as zwitterions, and arepreferred aspects of this invention.

Compounds of the formula (III) when in zwitterionic form tend to becrystalline, and as such are especially preferred aspects of thisinvention.

Compounds of this invention when in crystalline form may be solvated,for example hydrated.

The present invention provides a pharmaceutical composition whichcomprises a compound of this invention and a pharmaceutically acceptablecarrier.

The compositions of the invention include those in a form adapted fororal, topical or parenteral use and may be used for the treatment of theinfection in mammals including humans.

Suitable forms of the compositions of this invention include tablets,capsules, creams, syrups, suspensions, solutions, reconstitutablepowders and sterile forms suitable for injection or infusion. Suchcompositions may contain conventional pharmaceutically acceptablematerials such as diluents, binders, colours, flavours, preservatives,disintegrant and the like in accordance with conventional pharmaceuticalpractice in the manner well understood by those skilled in the art offormulating antibiotics.

Injectable or infusable compositions of a compound of the invention areparticularly suitable as high blood levels of the compound can occurafter administration by injection or infusion. Thus, one preferredcomposition aspect of this invention comprises a compound of theinvention in sterile form and most suitably in sterile crystalline form.The zwitterionic compounds of this invention are particularly suitablefor use in such compositions.

The injectable solution of the compound of this invention may be made upin a sterile pyrogen-free liquid such as water, aqueous ethanol or thelike.

An alternative approach to administering the compounds of this inventionand especially those zwitterionic compounds of the formula (II) is toutilise an injectable suspension. Such suspensions may be made up insterile water; sterile saline or the like and may also containsuspending agents such as polyvinylpyrrolidone, lecithin or the like(for example in the manner described for amoxycillin trihydrate inBelgian Pat. No. 839109). Alternatively such compositions may beprepared in an acceptable oil suspending agent such as arachis oil orits equivalent. The use of suspensions can give rise to advantageouslyprolonged blood levels of the medicament. Belgian Pat. No. 839109 may beconsulted for suitable methods and materials for producing injectableaqueous suspensions. For use in such suspensions the zwitterioniccompound of this invention should be in the form of fine particles asdescribed in said Belgian Patent.

Unit dose compositions comprising a compound of this invention adaptedfor oral administration form a further suitable composition aspect ofthis invention.

Unit dose compositions comprising a compound of this invention adaptedfor topical administration are also presented by this invention. In thisinstance `topical administration` also includes local administration tointernal surfaces of mammary glands of cattle, for example during thetreatment of mastitis by intra-mammary administration.

The compound of the formula may be present in the composition as soletherapeutic agent or it may be present together with other therapeuticagents such as a penicillin or cephalosporin. Considerable advantagesaccrue from the inclusion of a penicillin or cephalosporin since theresulting composition shows enhanced effectiveness (synergy).

Suitable penicillins for inclusion in the compositions of this inventioninclude benzylpenicillin, phenoxymethylpenicillin, carbenicillin,azidocillin, propicillin, ampicillin, amoxycillin, epicillin,ticarcillin, cyclacillin, pirbenicillin, azlocillin, mezlocillin,celbenicillin, and other known penicillins including pro-drugs thereforesuch as their in vivo hydrolysable esters such as the acetoxymethyl,pivaloyloxymethyl, α-ethoxycarbonyloxyethyl or phthalidyl esters ofampicillin, benzylpenicillin or amoxycillin, and aldehyde or ketoneadducts of penicillins containing a 6-α-aminoacetamide side chain (suchas hetacillin, metampicillin and analogous derivatives of amoxycillin)or α-esters of carbenicillin or ticarcillin such as their phenyl orindanyl α-esters.

Suitable cephalosporins for inclusion in the compositions of thisinvention include cefatrizine, cephaloridine, cephalothin, cefazolin,cephalexin, cephacetrile, cephamandole nafate, cephapirin, cephradine,4-hydroxycephalexin, cefaparole, cephaloglycin, and other knowncephalosporins or prodrugs thereof.

Such compounds are frequently used in the form of a salt or hydrate ofthe like.

Naturally if the penicillin or cephalosporin present in the compositionis not suitable for oral administration then the composition will beadapted for parenteral administration.

Highly favoured penicillins for use in the compositions of thisinvention include ampicillin, amoxycillin, carbenicillin andticarcillin. Such penicillins may be used as a pharmaceuticallyacceptable salt such as the sodium salt. Alternatively the ampicillin oramoxycillin may be used in the form of fine particles of thezwitterionic form (generally as ampicillin trihydrate or amoxycillintrihydrate) for use in an injectable suspension, for example, in themanner hereinbefore described for a compound of this invention.

The preferred penicillin for use in the synergistic composition isamoxycillin, for example as its sodium salt or trihydrate.

Particularly suitable cephalosporins for use in the compositions of thisinvention include cephaloridine and cefazolin which may be in the formof a pharmaceutically acceptable salt for example the sodium salt.

When present together with a cephalosporin or penicillin, the ratio of acompound of the invention to the penicillin or cephalosporin agent mayvary over a wide range of ratios, such as from 10:1 to 1:10 for exampleabout 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, (wt/wt, based on purefree antibiotic equivalent). Orally administrable compositionscontaining a compound of the invention will normally contain relativelymore synergist than corresponding injectable compositions.

The total quantity of a compound of the invention in any unit dosageform will normally be between 25 and 1000 mg and will usually be between50 and 500 mg, for example about 62.5, 100, 125, 150, 200 or 250 mg.

Compositions of this invention may be used for the treatment ofinfections of inter alia, the respiratory tract, the urinary tract andsoft tissues in humans and mastitis in cattle.

Normally between 50 and 1000 mg of the compounds of the invention willbe administered each day of treatment but more usually between 100 and750 mg of the compounds of the invention will be administered per day,for example at 1-6 doses, more usually as 2, 3 or 4 doses.

The penicillin or cephalosporin in the synergistic composition of thisinvention will normally be present at approximately the amount at whichit is conveniently used which will usually be expected to be from about62.5 to 1000 mg per dose, more usually about 125, 250 or 500 mg perdose.

One particularly favoured composition of this invention will containfrom 150 to 1000 mg of amoxycillin as the trihydrate or sodium salt andfrom 25 to 500 mg of a compound of this invention.

Most suitably this form of composition will contain a compound of theformula (II) when in crystalline zwitterionic form.

A further particularly favoured composition of this invention willcontain from 150 to 1000 mg of ampicillin or a pro-drug therefore andfrom 25 to 500 mg of a compound of this invention.

Most suitably this form of composition will contain ampicillintrihydrate, ampicillin anhydrate, sodium ampicillin, hetacillin,pivampicillinhydrochloride, bacampicillin hydrochloride, ortalampicillin hydrochloride. Most suitably this form of the compositionwill contain a compound of the formula (II) when in crystallinezwitterionic form.

Most suitably the preceding composition will contain from 200 to 700 mgof the penicillin component. Most suitably the preceding compositionwill comprise from 50 to 250 mg of a compound of the formula (II)preferably in crystalline zwitterionic form.

Such compositions may be adapted for oral or parenteral use except whencontaining an in vivo hydrolysable ester of ampicillin or amoxycillin inwhich case the compositions will not be adapted for parenteraladministration.

Another particularly favoured composition of this invention will containfrom 200 to 2000 mg of carbenicillin, ticarcillin or a pro-drugtherefore and from 50 to 500 mg of a compound of the invention.

Suitably this form of composition will contain di-sodium carbenicillin.Suitably this form of the composition will contain di-sodiumticarcillin.

More suitably this form of the composition will contain from 75 to 250mg of a compound of the formula (II) preferably in crystallinezwitterionic form. Such compositions containing di-salts ofcarbenicillin and ticarcillin will be adapted for parenteraladministration.

The present invention also provides a method of treating bacterialinfections in humans of domestic mammals which comprises theadministration of a composition of this invention.

Commonly the infection treated will be due to a strain of Staphylococcusaureus, Klebsiella aerogenes, Escherichia coli, Proteus sp. or the like.The organisms believed to be most readily treated by an antibacteriallyeffective amount of a compound of this invention is Staphylococcusaureus. The other organisms named are more readily treated by using asynergistically effective amount of the compound of the invention and apenicillin or cephalosporin. The administration of the two componentsmay take place separately but in general we prefer to use a compositioncontaining both the synergist and the penicillin or cephalosporin.

The indications for treatment include respiratory tract and urinarytract infections in humans and mastitis in cattle.

The present invention also provides a process for the preparation of acompound of formula (II) or a pharmaceutically acceptable salt or esterthereof which process comprises reacting a compound of formula (IV) or(V), or an ester thereof: ##STR6## wherein 2 is a displaceable group;with an amine of formula (VI): ##STR7## wherein A and X are as definedwith respect to formula (II) and R is a removable protecting group; andthereafter removing the group R.

Suitable groups Z include halogen, for example chlorine or bromine,alkyl- or aryl-sulphonyloxy, or acyloxy such as optionally substitutedC₁₋₆ alkanoyloxy for example acetoxy or dichloroacetoxy.

Suitably the reaction of the amine of the formula (VI) with the compoundof the formula (IV) or (V) will take place in an aprotic solvent such asacetonitrile or dimethylformamide at a non-extreme temperature, forexample -10° to +50°, more usually -5° to +25°, and conveniently withinthe range 0° to +20°.

Suitably the group R is one which may be removed by hydrogenation. Forexample R may be a group of the sub-formula (f):

    --CH.sub.2 CR.sub.7 ═CHR.sub.8                         (f)

wherein R₇ is a hydrogen atom or lower alkyl group and R₈ is a hydrogenatom, a lower alkyl group, or a phenyl group optionally substituted witha lower alkyl or lower alkoxy group; or R₇ and R₈ together represent abutadiene moiety.

Particularly suitable groups R include the following: CH₂ CH═CH₃, CH₂CH═CHC₆ H₅, CH₂ C(CH₃)═CH₂, CH₂ C(C₂ H₅)═CH₂, CH₂ C(nC₃ H₇)═CH₂, CH₂C(CH₃)═CHCH₃, CH₂ C(CH₃)═C(CH₃)₂, CH₂ C(CH₃)═CHC₂ H₅, CH₂ C(CH₃)═CHC₆ H₅and CH₂ C₆ H₅.

Favoured groups R are CH₂ CH═CHCH₃, CH₂ CH═CHC₆ H₅, CH₂ C(CH₃)═CH₂, CH₂C(CH₃)═CHC₆ H₅ and CH₂ C₆ H₅.

Particularly preferred groups R are CH₂ C(CH₃)═CH₂, CH₂ C(CH₃)═CHC₆ H₅and CH₂ C₆ H₅.

The group A and the ester group of compounds (IV) and (V) may be groupsthat can be removed after the reaction, for example by hydrogenation, oralternatively the ester groups may be left intact after the reaction.Thus four forms of compounds of this invention may be presented: (a)di-salts, (b) di-esters, (c) a mono-ester wherein group A is a hydrogenatom or a salifying group, and (d) a mono-ester wherein group A is theesterifying radical.

The hydrogenation is normally carried out in the presence of atransition metal catalyst.

The catalyst we have preferred to use is palladium, for example in theform of palladium on carbon (charcoal), palladium on barium sulphate,palladium on calcium carbonate, palladium black or the like.

A favoured catalyst is palladium on carbon (sometimes referred to aspalladium on charcoal); for example 5%, 10%, 20% or 30% palladium oncarbon.

A low, medium or high pressure of hydrogen may be used in this reaction,for example from 1 to 6 atomospheres.

The reaction is normally carried out at a non-extreme temperature, forexample from 0°-30° and more usually from 12°-25°. It is generallyconvenient to carry out the reaction at ambient temperature.

Suitably solvents for carrying out the hydrogenation include ethanol,n-propanol, isopropanol, tetrahydrofuran, dioxan, ethyl acetate ormixtures of such solvents or such solvents in the presence of water. Afavoured solvent is ethanol.

Favoured hydrogenolysable esters include benzyl and substituted benzylesters such as methoxybenzyl, nitrobenzyl (for example the p-nitrobenzylester), chlorobenzyl and bromobenzyl esters. A particularly suitablehydrogenolysable ester is the benzyl ester. A further particularlysuitable hydrogenolysable ester is the p-methoxybenzyl ester.

The product may generally be isolated from the reaction mixture byfiltering off the solids (the catalyst which should be well washed toremove the product) and then evaporating the solvent, preferably underlow pressure, to yield the initial product. Further purification may beeffected by such conventional methods as chromatography over celluloseor other mild stationary phase eluting with a C₁₋₄ alkanol optionally inthe presence of water and optionally in the presence of tetrahydrofuran.Evaporation of the combined active fraction (identified by aqueouspotassium permanganate spray on tlc) then yields the desired compound inpure form. The desired product is normally obtained in crystalline form(unless it is an unsalted ester). Trituration under ethanol, isopropanolor the like C₁₋₄ alkanol or other conventional solvent such as a ketone,either or ester solvent or other conventional solvent (for example of upto 6 carbon atoms and more suitably of up to 4 carbon atoms) may also beused to aid crystallisation. Recrystalisation from ethanol or the likemay also be employed. The solvent used in such processes mayadvantageously be moist.

Unsalted esters of the compounds of the formula (II) tend to be oils sothat it is often more convenient for handling to convert them into solidacid addition salts, for example by reaction with one equivalent of anacid.

The foregoing process is not preferred when preparing compounds of theformula (II) in which number of carbon atoms in the chain directlylinking the --NH-- group and the --CO₂ A group is one.

In an improved process for the preparation of compounds of the formula(II) wherein the number of carbon atoms in the chain directly linkingthe --NH-- group and the CO₂ A group is one, the present inventionprovides a process which comprises the reduction with a complex hydrideof a salt of a compound of the formula (VII): ##STR8## wherein Ar is aphenyl group optionally substituted by a bromine, fluorine or chlorineatom or an alkyl group of 1-3 carbon atoms or an alkoxy group of 1-3carbon atoms and A is as defined in relation to formula (II).

Most suitably the complex hydride is a water soluble complex hydride.

Suitable water soluble complex hydrides include borohydrides such aslithium borohydride, sodium borohydride, potassium borohydride or thelike. In general an excess of the hydride is employed.

Suitably the reaction is carried out in an aqueous medium, for examplein water or in a mixture of water with an inert water miscible organicsolvent such as tetrahydrofuran, dioxan or the like.

It is a favoured feature of this invention that ambient and near ambienttemperatures may be employed, for example the reaction may be carriedout at a temperature of from 0°-30° and conveniently at ambient, forexample at about 18°-25°.

The pH of the reaction is best kept below 10 and this may be effected bythe addition of an acid such as hydrochloric or like mineral acidsimultaneously with the complex hydride. This may be carried out in apH-stat or other similar system.

Once the reaction is over it is advantageous to return the pH to about5-8.

The desired product may be obtained from the reaction mixture byevaporation of the solvent. Purification may be effected bycrystallisation (for example before all the solvent has been evaporatedoff) or by column chromatography, for example using silica gel orcellulose and butanol/ethanol/water 4/4/1.

The compounds of the formula (VII) are novel and as such form an aspectof this invention.

The present invention also provides a process for the preparation of acompound of the formula (VII) which process comprises the reaction of9-aminodeoxyclavulanic acid with a compound of the formula (VIII):

    ArCOCO.sub.2 A                                             (VIII)

wherein Ar is as defined in relation to formula (VII) and A is asdefined in relation to formula (II) in an aqueous solvent wherein thesolution is maintained at an alkaline pH.

The pH of the solution is most suitably maintained in the region of 7-10and preferably 8-9. This may be effected by the addition of base such asan alkali or alkaline earth metal hydroxide, a carbonate of bicarbonateor with a strong organic base which is unreactive towards aldehydes.Thus suitable bases include lithium hydroxide, sodium hydroxide,potassium hydroxide, calcium hydroxide, barium hydroxide, sodiumcarbonate, potassium bicarbonate, triethylamine and the like. It isconvenient to add the base automatically, for example in a pH-stat.

The present invention also provides a process for the preparation of anester of a compound of the formula (II) which process comprises thereaction of the compound of the formula (II) with an esterifying agent.

The zwitterionic compound of the formula (II) may be dissolved orsuspended in a solvent such as dimethylformamide,hexamethylphosphoramide, dichloromethane, ethyl acetate or othernon-esterifiable solvents and therein esterified. Suitable temperaturesfor such a reaction range from about 0° to about 25°. Suitableesterifying reagents include reactive halides and their equivalents,alkyl oxonium salts and the like.

When a reagent such as a reactive iodide, chloride, bromide, tosylate,mesylate or the equivalent is used, the resulting salt is generallysuitable for use in a composition of this invention. Alternatively, thesalt may be converted to a free base or alternative salt. When an alkyloxonium salt is used, it is preferred to convert the resultingtetrafluoroborate to the free base or alternative salt. The variousaformentioned salts may be converted to the free base by neutralisation,for example by contacting a solution of the salt in water with anorganic phase, neutralising the salt by adding a base and extracting theliberated amine into the organic phase. This amine may thereafter bere-salted by reacting with an appropriate acid, for example in a dryorganic solvent. It is generally preferred to use not more than oneequivalent of acid for this process. Alternatively the originally formedsalt may be converted into the alternative salt using an ion exchangematerial, for example, by passing an aqueous solution of one saltthrough a bed of an anion exchange resin in the form of the desired saltsuch as the chloride form.

The salts may normally be obtained in solid form by dissolving in afairly polar organic solvent (such as ethanol, tetrahydrofuran or thelike) and then precipitating using a non-polar solvent such as diethylether, cyclohexane or the like.

The salts of the esters of the compounds of the formula (II) maynormally be obtained in crystalline form by conventional methods such astrituration under (or crystallisation or recrystallisation from) asuitable organic solvent such as ether, acetone, acetonitrile,tetrahydrofuran or the like.

The present invention also provides a process for the preparation of anester of the compound of the formula (II) which process comprises thereaction of an acid addition salt of the compound of the formula (II)with an alcohol in the presence of a condensation promoting agent.

Suitable condensation promoting agents for use in this process includecarbodiimides such as dicyclohexylcarbodiimide and the chemicalequivalents thereof.

The acid addition salt may be formed in situ or may be preformed. Theacid employed will normally be a strong acid such as a methane sulphonicacid, p-toluene sulphonic acid or the like or trifluoroacetic acid orthe like.

The reaction is normally carried out in an inert organic solvent. Whenthe ester being formed is that of a liquid alcohol it is convenient touse that alcohol as the solvent or as part of the solvent system. Theesterification is generally performed at a no-extreme temperature suchas 0°-35°, for example from about 10°-25° C. Conveniently the reactionmixture may be performed at ambient temperature.

Other methods of preparing esters of the compounds of the formula (II)are those described in the aforementioned patents and applications whichare incorporated herein by reference.

In a further aspect this invention also provides a process for thepreparation of compounds of formula (II) which process comprisesreacting a compound of formula (IX) or an ester thereof: ##STR9## whereR^(a) represents hydrogen or a removable protecting group; with acompound of formula (X):

    Z.sup.1 --X--CO.sub.2 A                                    (X)

wherein X and A are as defined with respect to formula (II) and Z^(a) isa readily displaceable group; and thereafter removing any group R^(a)which is not hydrogen.

When the group R^(a) is not hydrogen it may conveniently be any of thegroups R defined above with respect to formula (VI).

Suitable groups Z^(a) include halogen, alkyl- or aryl-sulphonyloxy, suchas methanesulphonyloxy, benzenesulphonyloxy, p-toluenesulphonyloxy,p-bromobenzenesulphonyloxy. A preferred group Z^(a) is iodine. When X isCH₂, Z^(a) is suitably bromine.

The reaction of compound (IX) with compound (X) is conveniently carriedout in an inert organic solvent such as dimethylformamide, acetonitrileor methylene dichloride, preferably in the presence of a strongnon-nucleophilic organic base, and at a non-extreme temperature forexample -10° to +50°, more usually -5° to +20° and conveniently in therange -5° to +10°.

The foregoing process is not preferred when preparing compounds offormula (II) in which X represents --CH₂ --CH₂ --.

The following Examples illustrate the preparation of compounds of thisinvention.

EXAMPLE 1 (a) Benzyl9-N-[2'-(benzyloxycarbonyl)ethyl]-N-(2"-methyl-3"-phenylallyl)aminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (1.01 g:2.53 mM) in drydimethylformamide (30 cm³) at -15° was treated with 1.9 equivalents ofN-[2-(benzyloxycarbonyl)ethyl]-N-(2'-methyl-3'-phenylallyl)amine andstirred at -15° to 0° over 2 hours. The mixture was poured into ethylacetate (300 cm³), washed with water (5×100 cm³), saturated brine (5×100cm³), dried (anhydrous magnesium sulphate) and evaporated to an oil.This oil was chromatographed on silica eluting with methylacetate:cyclohexane (1:2). Fractions were collected containing the titlecompound Rf (SiO₂ /ethyl acetate:cyclohexane (1:1)=0.83 (detection byaqueous potassium permanganate spray), combined fractions wereevaporated to yield 0.52 g of an oil (35%). (film) 1805, 1740, 1700,1305, 1180, 1015, 755, 700 cm⁻¹ ; (CDCl₃) 1.78 (3H, s), 2.35-2.80 (4H,m), 2.93 (1H, d, J 17 Hz), 2.96 (2H, s), 3.18 (2H, d, J 7 Hz), 3.37 (1H,dd, J 17 and 3 Hz), 4.69 (1H, 6, J 7 Hz), 5.08 (3H, broad s), 5.16 (2H,s), 5.60 (1H, broad d, J 3 Hz), 6.34 (1H, broad s), 7.23 (5H) and 7.30(10H) (2×s).

(b) 9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid

Benzyl9-N-[2'-(benzyloxycarbonyl)ethyl]-N-(2"-methyl-3"-phenylallyl)aminodeoxyclavulanate(134 mg: 0.23 mM) in ethanol:tetrahydrofuran (15 cm³ ; 1:1) washydrogenolysed at atmospheric pressure in the presence of 10% palladiumon carbon (45 mg, prehydrogenated for 15 minutes) for 20 minutes, whenwater (3 cm³) was added and hydrogenolysis continued for 1 hour. Thecatalyst was filtered off and washed with ethanol (20 cm³). The filtratewas evaporated to an oil, on addition of methanol crystals formed whichwere filtered off cold and washed with a little cold methanol. Drying invacuo afforded the title compound as a white crystalline solid, yield=26mg (42%), rF (SiO₂ /butanol:propan-2-ol:water; 7:7:6)=0.38.

(Nujol) (3700-2000), (1805-1795), 1720, 1700, 1612, 1580, 1305, 1230,1190, 1122, 1075, 1045, 1020, 1005, 995, 920, 895, 805, 785, 755 cm⁻¹ ;(KBr) (3700-3250), (3250-2890), (2890-2500), (2500-2200), 1790 broad,1715 broad, 1575 very broad), 1400, 1300, 1190, 1118, 1082, 1070, 1045,1015, 915, 895, 795, 758 cm⁻¹ ; (D₂ O) 2.5502.87 (2H, m), 3.10 (1H, d, J17 Hz), 3.05-3.38 (2H, m), 3.58 (1H, dd, J 17 and 3 Hz), 3.75 (2H, d, J7 Hz), 4.81 (1H, dt, J 7 Hz and 1.25 Hz), 5.03 (1H, broad s), 5.75 (1H,d, J 3 Hz).

EXAMPLE 2 (a) Benzyl9-N-[2'(benzyloxycarbonyl)ethyl]-N-(2"-Methylallyl)aminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (0.98 g:2.45 mM) in drydimethylformamide (50 cm³) at -10° was treatedwith 1.9 equivalents ofN-[(2-benzyloxycarbonyl)ethyl]-N-(2'-methylallyl)amine with stirring.The mixture was stirred from -10° to 0° over 1 hour then at 0° for 11/2hours. The mixture was poured into ethyl acetate (250 cm³), washed withwater (6×100 cm³) saturated brine (6×150 cm³), dried (anhydrousmagnesium sulphate) and evaporated to an oil. This oil waschromatographed on silica eluting with ethyl acetate:cyclohexane (1:3).Fractions were collected containing the title compound and the combinedfractions were evaporated to yield the title compound as an oil (250 mg)(20%), (film) 1802, 1740, 1700, 740, 700 cm⁻¹.

(b) 9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid

Benzyl9-N-[(2'-benzyloxycarbonyl)ethyl]-N-(2"-methylallyl)aminodeoxyclavulanate(0.23 g:0.45 mM) in tetrahydrofuran:ethanol (1:2; 25.cm³) washydrogenolysed in the presence of 10% palladium on carbon (90 mg; whichhad been prehydrogenated for 15 minutes) for 15 minutes when water (3cm³) was added and hydrogenolysis continued for 3/4 hour. The catalystwas filtered off and washed with aqueous ethanol (20 cm³), the clearfiltrate was evaporated and methanol (5 cm³) added, the resultingcrystals were filtered off (0° C.) and washed with cold methanol, dryingafforded 27 mg (22%) of the title compound as a white crystalline solid,Ff (SiO₂ /ethanol:water:ethyl acetate; 2:2:5)=0.26. (Nujol) (3700-2200)very broad 1800, 1720, 1695, 1605, 1575, 1300, 1227, 1187, 1042, 1015,915, 892 cm⁻¹. The proton magnetic resonance spectrum was consistentwith the required product.

EXAMPLE 3 (a)N-[4-(Benzyloxycarbonyl)butyl]-N-(2-methyl-3-phenylallyl)amine

13.7 g (36 mM) of 4-(benzyloxycarbonyl)butylamine para toluenesulphonate salt was stirred vigorously in ethyl acetate-water whilstbeing treated with aqueous sodium hydroxide to pH 9.5. The organic phasewas washed with saturated brine, dried (anhydrous magnesium sulphate),benzyl alcohol (100 cm³) added, and the ethyl acetate evaporated. Thesolution was treated with one equivalent of α-methylcinnamaldehyde andstirred for 1/2 hour. Dichloromethane (50 cm³) was added and thereaction mixture treated with excess sodium borohydride. The mixture wasfiltered and the filtrate diluted with dichloromethane (100 cm³), washedwith saturated brine (4×150 cm³) and dried (anhydrous magnesiumsulphate). The dichloromethane was removed by evaporation under reducedpressure and the residue diluted with diethyl ether (300 cm³). Thissolution was treated with excess para toluene sulphonic acid in ether.The resultant crystals were filtered off, washed with ether and dried toyield 2.7 g (15%) of the title compound as a para-toluene sulphonatesalt. This salt was extracted into ethyl acetate with aqueous sodiumhydroxide (pH 9-10). The organic phase was washed with saturated brine,dried (anhydrous magnesium sulphate) and evaporated to an oil,yield=1.85 g (15%), (film) 3330, 1735, 1450, 1155, 750, 700 cm⁻¹ ;(CDCl₃) 1.35-2.00 (5H, m), 1.85 (3H, d, J 1 Hz), 2.37 and 2.62 (4H, 2 xt, J 6.5 Hz), 3.27 (2H, s), 5.10 (2H, s), 6.42 (1H, broad s), 7.26 and7.34 (10H, 2×s).

(b) Benzyl9-N-[4'-(benzyloxycarbonyl)butyl]-N-(2"-methyl-3"-phenylallyl)aminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (10 g: 2.5 mM) in dry dimethylformamide(20 cm³) was treated at -15° with 1.9 equivalent ofN-[4-(benzyloxycarbonyl)butyl]-N-(2-methyl-3-phenylallyl)amine, andstirred between -10° and -5° over 11/2 hours. The reaction mixture waspoured into ethyl acetate (200 cm³), washed with water (5×150 cm³),saturated brine (5×150 cm³), dried (anhydrous magnesium sulphate) andevaporated in the presence of toluene to a small volume. This crudeproduct was chromatographed on silica eluting with ethylacetate:cyclohexane (1:3), fractions were collected containing the titlecompound, Rf (SiO₂ /ethyl acetate:cyclohexane; 1:2)=0.5 (detection byaqueous potassium permanganate spray). Combined fractions wereevaporated to yield the title compound as an oil, 0.21 (14%), (film)1805, 1740, 1700 (shoulder), 745, 705 cm⁻¹.

The proton magnetic resonance spectrum was consistent with the desiredproduct.

(c) 9-N-(4'-Carboxybutyl)aminodeoxyclavulanic acid

Benzyl9-N-[4'-benzyloxycarbonyl)butyl]-N-(2"-methyl-3"-phenylallyl)aminodeoxyclavulanate(200 mg; 0.33 mM) in ethanol-tetrahydrofuran; 20 cm³ (10:1) plus water(1/2 cm³) was hydrogenolysed at atmospheric pressure in the presence of10% palladium on carbon (80 mg; which had been prehydrogenated for 10minutes) for 11/2 hours. The catalyst was filtered off and washed withethanol (20 cm³), then with aqueous ethanol (50 cm³), the aqueouswashings were collected separately and were evaporated to afford thetitle compound as a white solid, this solid was washed with coldmethanol and dried to yield 16.4 mg. The ethanolic washings from thecatalyst were hydrogenolysed for a further 21/4 hours in the presence of10% palladium on carbon (50 mg). The catalyst was filtered off andwashed with aqueous ethanol (30 cm³), the filtrate was evaporated to anoil, methanol was added (5 cm³) and the solution cooled (0°). Theresultant white crystals were filtered off and washed with cold (0°)methanol and dried to afford a further 7.8 mg of the title compound.Total yield=24.2 mg (25%), Rf (SiO₂ /ethylacetate-ethanol-water;5:2:2)=0.25, ν (Nujol) 1805, 1725, 1695, 1610, 1585, 1300, 1185, 1045,1015, 892 cm⁻¹ ; δ(D₂ O) 1.35-1.85 (4H, m), 2.13-2.50 (2H, m), 2.78- 3.1(2H, m), 3.08 (1H, d, J 17 Hz), 3.55 (1H, dd, J 17 and 3 Hz), 3.68 (2H,d, J 8 Hz), 4.77 (1H, broad t, J 8 Hz), 4.98 (1H, s), 5.73 (1H, d, J 3Hz).

EXAMPLE 4 (a) N-[4-(Benzyloxycarbonyl)benzyl]-N-(2'-methylallyl)amine

4-(Benzyloxycarbonyl)benzaldehyde (12 g; 50 mM) in benzyl alcohol (70cm³) was treated with 2-methylallylamine (4.5 cm³ ; 1 equivalent) andstirred for 3 hours. Dichloromethane (50 cm³) and water (2 cm³) wereadded followed by excess sodium borohydride. The precipitated solid wasfiltered off and the filtrate diluted with dichloromethane (200 cm³).This solution was washed with brine (4×200 cm³) and dried (anhydrousmagnesium sulphate). The dichloromethane was evaporated and diethylether added (300 cm³), to this solution was added para-toluene sulphonicacid in ether until the solution was acidic. The resultant white solidwas filtered off, washed with ether and dried to yield 16.3 g of thepara-toluene sulphonate salt. This salt was stirred vigorously withwater and ethyl acetate whilst being treated with aqueous sodiumhydroxide to pH 9.00. The ethyl acetate phase was washed with water(3×100 cm³), saturated brine (4×100 cm³), dried (anhydrous magnesiumsulphate) and evaporated to an oil. This oil was chromatographed onsilica eluting with ethyl acetatecyclohexane (1:3). Fractions werecollected containing the title compound and combined fractions wereevaporated to yield a viscous oil, 8.69 g (60%), ν (film) 3340 (broad),1720, 1610, 1450, 1270, 1175, 1095, 1015, 895, 750, 695 cm⁻¹ ; δ (CDCl₃)1.60 (1H, s, exchanges with D₂ O), 1.78 (3H, s), 3.17 (2H, s), 3.81 (2H,s), 4.93 (2H, broad s), 5.37 (2H, s), 7.29-7.60 (7H, m), 8.10 (2H,d_(AB) J 8 Hz).

(b) Benzyl9-N-[4'-(benzyloxycarbonyl)benzyl]-N-(2"-methylallyl)aminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (2.73 g; 6.83 mM) in drydimethylformamide (20 cm³) at 0° was treated with 1.9 equivalents ofN-[4-(benzyloxycarbonyl)benzyl]-N-(2'-methylallyl)amine indimethylformamide (20 cm³) dropwise over ten minutes, then stirred at 0°for 4 hours and allowed to warm up to room temperature over 3/4 hour.The mixture was poured into ethyl acetate (250 cm³), washed with water(6×150 cm³), saturated brine (6×150 cm³), dried (MgSO₄) and evaporatedto an oil. This oil was chromatographed on silica, eluting with ethylacetate-cyclohexane (1:3). Fractions were collected containing the titlecompound Rf (SiO₂ /ethyl acetatecyclohexane; 1:1)=0.86 (detection byaqueous potassium permanganate spray). Combined fractions wereevaporated to an oil, yield=0.8 g (21%), ν (film) 1805, 1750, 1720, 895,760, 750, 700 cm⁻¹ ; δ (CDCl₃) 1.70 (3H, s), 2.85 (2H, s), 6βH obscured,3.09 (2H, d, J 7 Hz), 3.43 (1H, dd, J 17 and 3 Hz), 3.45 (2H, s), 4.71(1H, t, J 7 Hz), 4.85 (2H, broad s), 5.05 (1H, s), 5.17 (2H, s), 5.35(2H, s), 5.60 (1H, d, J 3 Hz), 7.20-7.55 (12H, m), 8.00 (2H, d_(AB) J 8Hz).

(c) 9-N-(4'-Carboxybenzyl)aminodeoxyclavulanic acid

Benzyl9-N-[4'-(benzyloxycarbonyl)benzyl]-N-(2"-methylallyl)aminodeoxyclavulanate(0.71 g; 1.25 mM), in tetrahydrofuran-ethanol (1:1; 20 cm³) and water (1cm³) was hydrogenolysed at atmospheric pressure in the presence of 10%palladium on carbon (200 mg) (which had been prehydrogenated for 15minutes) for 1 hour. The catalyst was filtered off, washed with ethanol(20 cm³), aqueous ethanol (250 cm³) and aqueousisopropanoltetrahydrofuran (150 cm³) until the filtrate no longer showedany product present by thin layer chromatography. These aqueous washingswere collected separately and were evaporated to give a whitecrystalline solid. This solid was washed with ethanol and dried to yield183 mg of the title compound, Rf (SiO₂ /ethylacetate-ethanol-water;5:2:2)=0.32 (detection by aqueous potassium permanganate spray). ν(Nujol) 1805, 1690, 1602, 1578, 1298, 1275, 1185, 1122, 1150, 1118, 990,945, 890, 860, 755, 705 cm⁻¹ ; ν (KBr) (3700-2200, broad multiplepeaks), 1808, 1690, 1600, 1580, 1465, 1404, 1300, 1275, 1187, 1127,1055, 1022, 993, 947, 895, 867, 760, 712 cm⁻¹ ; δ (D₂ O/pyridine d-5)3.15 (1H, d, J 17 Hz), 3.80 (1H, broad d, J 17 Hz), 4.07 (2H, d, J 8Hz), 4.58 (2H, s), 8CH and 3CH obscured by HOD, 6.20 (1H, broad s), 7.85(2H, d_(AB), J 8 Hz), 8.45 (2H, d_(AB), J 8 Hz).

EXAMPLE 5 9-(DL-α-Carboxybenzylamino)deoxyclavulanic acid

A solution containing 9-aminodeoxyclavulanic acid (0.2 g) and methylbenzoylformate (0.8 g) in water (10 ml) and tetrahydrofuran (15 ml) wasstirred and maintained at pH 8-9 by the automatic addition of 1M LiOHsolution. Uptake of LiOH solution was continous, and was allowed toexceed the amount required for Schiff's base formation by a considerableamount (˜2 fold). Sodium borohydride (0.15 g) was then addedconcurrently with HCl to keep the pH below 9. Once the addition wascomplete the reaction mixture was brought to pH 7 by the addition ofdilute HCl. The reaction mixture was evaporated to dryness and extractedwith ethyl acetate. The insoluble residue was re-evaporated with 1.5 mlwater, then subjected to column chromatography on silica gel usingn-butanol-ethanol-water 7:7:6 v/v elution solvent. Fractions containingthe desired product were combined and evaporated to dryness in vacuo.The residue was extracted twice with small volumes of acetone, then withether and finally dried in vacuo, to yield the product as a pale buffsolid; ν (Nujol) 2500-3700 (broad) 1785, 1700, and 1620 cm⁻¹ (broad)δ(D₂ O) 3.20 (1H, d, J 17 Hz), 3.66 (1H, dd, J 17 and 3 Hz), 3.74 (2H,d, J 8 Hz), (HOD at 4.7 obscured many peaks) 5.85 (1H, d, J 3 Hz), and7.50 (5H, s).

Tlc ran in the elution solvent above showed the product to be quitedistinct from 9-aminodeoxyclavulanic acid.

EXAMPLE 6 (a) Benzyl9-N-(2'-methoxycarbonylethyl)-N-benzylaminodeoxyclavulanate

Benzyl 9-O-dichloroacetylclavulanate (5.3 g; 13.3 mmol), in drydimethylformamide (70 cm³) at -15° C. was treated dropwise withN-benzyl-O-methyl-β-alanine (1.9 equivalents) in dimethylformamide (20cm³). The reaction mixture was stirred between -10° and +10° over 11/2hours. The reaction mixture was poured into methyl acetate (300 cm³),dried (anhydrous magnesium sulphate) and evaporated to oil. This oil waschromatographed on silica eluting with methyl acetate:toluene; 1:4.Fractions were collected containing the title compound Rf (SiO₂ /methylacetate:toluene; 1:4)=0.4 (detecting by aqueous potassium permanganatespray), combined fractions were evaporated to afford an oil, yield=1.86g (30%); ν (film) 1805, 1740, 1695, 745, 700 cm⁻¹, δ (CDCl₃) 2.28-2.82(4H, m, NCH₂ CH₂ CO₂), 2.96 (1H, d, J 17 Hz, 6βCH), 3.05 (2H, d, J 7 Hz,9CH₂), 3.41 (1H, dd, J 17 and 3 Hz, 6αCH), 3.45 (2H, s, NCH₂ C₆ H₅),3.59 (3H, s, CO₂ CH₃), 4.69 (1H, bt, J 7 Hz, 8CH), 5.04 (1H, bs, 3CH),5.15 (2H, s, CO₂ CH₂ C₆ H₅), 7.22 and 7.30 (2×5H, 2×s, 2×CH₂ C₆ H₅).

(b) 9-N-(2'-Methoxycarbonylethyl)aminodeoxyclavulanic acid

Benzyl 9-N-(2'methoxycarbonylethyl)-N-benzylaminodeoxyclavulanate (1.7g; 3.66 mmol) in ethanol (25 cm³), tetrahydrofuran (15 cm³) and water (5cm³) was hydrogenolysed at atmospheric pressure in the presence of 10%palladium on carbon (0.5 g; which had been prehydrogenated for 10minutes) for 1.3 hours. The catalyst was filtered off and washed withethanol (20 cm³) then with aqueous ethanol (100 cm³), this aqueous washwas evaporated to afford a white crystalline solid, the solid was washedwith cold ethanol and dried to give 0.66 g of the title compound. Theethanolic catalyst washings were evaporated to an oil, ethanol was addedand cooled, crystals formed slowly which after washing with ethanol anddrying afforded a further 35 mg of the title compound; total yield=0.70g (67%) Rf (SiO₂ /ethyl acetate:ethanol:H₂ O; 5:3:3)=0.45 (detection byaqueous potassium permanganate spray). (D₂ O) CH₃ CN 2.00 as internalstandard; 2.78 (2H, t, J 6 Hz, CH₂ CO₂), 3.08 (1H, d, J 17 Hz, 6βCH),3.25 (2H, 6, NCH₂ CH₂, J 6 Hz), 3.56 (1H, dd, J 17 and 3 Hz, 6αCH), 3.67(3H, s, CO₂ CH₃), 3.74 (2H, d, J 7.5 Hz, 9CH₂), 4.78 (1H, dt, J 7.5 and1 Hz, 8CH), 4.97 (1H, bs, 3CH), 5.75 (1H, d, J 3 Hz, 5αCH). (KBr) 1805,1740, 1700, 1610, 1575, 1430, 1404, 1378, 1315, 1305, 1223, 1205, 1192,1125, 1075, 1043, 1025, 1005, 920, 896, 815, 792, 769 cm⁻¹.

EXAMPLE 7 (a) N,O-Dibenzyl β-alanine

β-Alanine benzyl ester (7.3 g; 45 mmol) was dissolved in chloroform (150cm³) and treated with benzaldehyde (1.2 equivalents) and stirred for11/2 hours. Benzyl alcohol (5 cm³) was added followed by excess sodiumborohydride. The reaction mixture was filtered and washed with saturatedbrine (3×200 cm³), dried (anhydrous magnesium sulphate) and evaporatedto an oil. This oil was dissolved in ether (300 cm³) and treated withexcess para-toluene sulphonic acid in ether. The resultant white solidwas filtered off, washed with dry ether and dried to afford 12.83 g(65%) of N,O-dibenzyl β-alanine p-toluene sulphonate. This salt wasstirred vigorously in ethyl acetate (150 cm³) and water (150 cm³) whilstbeing treated with sodium hydroxide solution to pH 10. The ethyl acetatephase was washed with saturated brine (5×200 cm³), dried (anhydrousmagnesium sulphate) and evaporated to afford 7.3 g (60%) of the titlecompound as an oil. (film) 3320, 1746, 1495, 1452, 1382, 1350, 1168,740, 700 cm⁻¹. (CDCl₃) 1.81 (1H, s, exchanges with D₂ O, NH), 2.4-3.1(4H, m, NCH), 5.12 (2H, s, OCH₂ C₆ H₅), 7.29 and 7.34 (2×5H, 2×s, 2×CH₂C₆ H₅), C₁₇ H₁₈ NO₂ (M⁺ -H) requires 268.1336; 268.1355; found, m/e 270(M⁺ +H), 269 (M⁺), 268 (M⁺ -H), 178 (M⁺ -91), 120, 118, 107, 106, 92,91, 65.

(b) Benzyl 9-N-(2'-benzyloxycarbonylethyl)-N-benzylaminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (5.24 g; 13.1 mmol) in drydimethylformamide (50 cm³) at -10° C. was treated with 1.9 equivalentsof N,O-dibenzyl βalanine (6.7 g) in dimethylformamide (20 cm³), dropwiseover 10 minutes, then stirred for 11/4 hours between 0° and +10°. Thereaction mixture was poured into ethyl acetate (250 cm³) and washed withwater (4×200 cm³) and saturated brine (4×200 cm³), dried (anhydrousmagnesium sulphate) and evaporated to an oil. This oil waschromatographed on silica eluting with toluene-ethyl acetate; 4:1.Fractions were collected containing the title compound, Rf (SiO₂/toluene:ethyl acetate; 4:1)=0.6 (detection by aqueous potassiumpermanganate spray). Combined fractions were evaporated to afford thetitle compound as an oil 2.9 g (41%). (film) 1805, 1740, 1700, 740, 700cm⁻¹, (CDCl₃) 2.36-2.90 (4H, brm, NCH₂ CH₂ CO₂), 2.93 (1H, d, J 17 Hz,6βCH), 3.15 (2 H, d, J 7 Hz, 9CH₂), 3.38 (1H, dd, J 17 and 3 Hz, 6αCH),3.45 (2H, s, NCH₂ C₆ H₅), 4.68 (1H, bt, J 7 Hz, 8CH), 5.02 (1H, s, 3CH),5.07, 5.15 (2H×2H, 2×s, 2×CO₂ CH₂ C₆ H₅), 5.58 (1H, d, J 3 Hz, 5αCH),[7.22 (5H, s), 7.30 (10H, s), 3×CH₂ C₆ H₅ ].

(c) 9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid

Benzyl 9-N-(2'-benzyloxycarbonylethyl)-N-benzylaminodeoxyclavulanate2.88 g; 5.33 mmol) in ethanol (30 cm³), tetrahydrofuran (20 cm³) andwater (5 cm³) was hydrogenolysed for 1 hour in the presence of 0.9 g of10% palladium on carbon. The reaction mixture was filtered and thesolids washed with ethanol (20 cm³), then with aqueous ethanol (300cm³). The aqueous washings were evaporated to afford a white crystallinesolid, this solid was slurried in cold methanol and filtered, washedwith cold methanol and dried to afford 0.70 g (49%) of the titlecompound. The ethanolic catalyst washings were evaporated to an oil,redissolved in aqueous methanol (30 cm³) (H₂ O:MeOH; 1:6) andrehydrogenolysed with 100 mg of fresh palladium on carbon for 11/2hours. The solids were filtered off, washed with ethanol (20 cm³) thenwith water (100 cm³), the water washings were evaporated, methanol added(30 cm³) and cooled, the resultant crystals were filtered off, washedwith cold methanol and dried to afford a further 140 cm (9.7%) of thetitle compound. Total yield 0.84 g (58%), (Nujol) 1810, 1730, 1705,(1680-1505) cm⁻¹, the proton magnetic resonance spectrum was consistantwith the desired compound.

EXAMPLE 8 (a) Methyl9-N-(2-benzyloxycarbonylethyl)-N-benzylaminodeoxyclavulanate

Methyl dichloroacetylclavulanate (4.5 g; 13.9 mmol) in drydimethylformamide (50 cm³) at -10° was treated with 1.9 equivalents ofN,O-dibenzyl β-alanine (7.1 g) and stirred for 23/4 hours between -10°and +20°. The mixture was poured into ethyl acetate (300 cm³) and washedwith water (5×100 cm³) and saturated brine (5×100 cm³), dried (anhydrousmagnesium sulphate) and evaporated to an oil. This crude product waschromatographed on silica eluting with toluene:ethyl acetate; 4:1,fractions were collected containing the title compound Rf (SiO₂/toluene-ethyl acetate; 4:1)=0.5 detection by aqueous potassiumpermanganate spray), combined fractions were evaporated to afford anoil, yield=0.97 (15%) (film) 1805, 1750, 1700, 1495, 1455, 1440, 1310,1240, 1180, 1120, 1010, 740, 700 cm⁻¹. (CDCl₃) 2.36-2.95 (4H, m, NCH₂CH₂ CO₂), 2.95 (1H, d, J 17 Hz, 6βCH), 3.17 (2H, d, J 7 Hz, 9CH₂), 3.40(1H, dd, J 17 and 3 Hz, 6αCH), 3.53 (2H, s, NCH₂ C₆ H₅), 3.72 (3H, s,CO₂ CH₃), 4.70 (1H, t, J 7 Hz, 8CH), 5.00 (1H, s, 3CH), 5.08 (2H, s, CO₂CH₂ C₆ H₅), 5.60 (1H, d, J 3 Hz, 5αCH), 7.23 and 7.23 (2×5H, 2×s, 2×CH₂C₆ H₅).

(b) Methyl 9-N-(2'-carboxyethyl)aminodeoxyclavulanate

Methyl 9-N-(2'-benzyloxycarbonylethyl)-N-benzylaminodeoxyclavulanate(0.87 g; 1.88 mmol) in ethanol (15 cm³), tetrahydrofuran (10 cm³) andwater (5 cm³) was hydrogenolysed at atmospheric pressure in the presenceof 10% palladium on carbon (260 mg; which had been prehydrogenated for15 minutes), for 41/4 hours. The catalyst was filtered off and thefiltrate evaporated to a foam, yield=1.1 g. 0.6 g of this crude productwas chromatographed on silica eluting with ethyl acetate-ethanol-water(5:4:3), fractions were collected containing the title compound Rf (SiO₂/ethyl acetate-ethanol-water; 5:2:2)=0.33 (detection by aqueouspotassium permanganate spray). Combined fractions were evaporated toafford 200 mg of a foam. (KBr) 1800, 1745, 1700, 1625 (shoulder), 1590cm⁻¹.

EXAMPLE 9 (a) Benzyl9-N-(5'-benzyloxycarbonylpentyl)-N-benzylaminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (8.12 g; 20.3 mmol) in drydimethylformamide (70 cm³) at -15° was treated with 1.9 equivalents ofN-benzyl(5'-benzyloxycarbonylpentyl) amine dropwise in dimethylformamideand stirred for 35 minutes between -15° and -10°. The mixture was pouredinto ethyl acetate (300 cm³) and was washed with water (4×100 cm³) andsaturated brine (6×100 cm³) dried and evaporated in the presence oftoluene to low volume. This crude product was chromatographed on silicaeluting with toluene-ethyl acetate (4:1). Fractions were collectedcontaining the title compound Rf (SiO₂ /toluene-ethyl acetate; 4:1)=0.5(detection by aqueous potassium permanganate spray), combined fractionswere evaporated to an oil, yield=1.5 g (13%), (film) 1805, 1740, 1700,1498, 1455, 1305, 1172, 1015, 740, 700 cm⁻¹.

(b) 9-N-(5'-Carboxypentyl)aminodeoxyclavulanic acid

Benzyl 9-N-(5'-benzyloxycarbonylpentyl)-N-benzylaminodeoxyclavulanate(1.43 g; 2.45 mmol) in ethanol (20 cm³), tetrahydrofuran (10 cm³) andwater (4 cm³) was hydrogenolysed in the presence of 10% palladium oncarbon (400 mg which had been prehydrogenated for 10 minutes) for 21hours at atmospheric pressure. The catalyst was filtered off and washedwith ethanol (20 cm³) then with aqueous ethanol (100 cm³), this aqueouswashing was evaporated to afford a white crystalline solid which waswashed with ice cold ethanol and dried to give 265 mg of the titlecompound, Rf (SiO₂ /ethyl acetate-ethanol-water; 5:3:3)=0.55 (detectionby aqueous potassium permanganate spray). The initial filtrate andethanolic washings were evaporated to afford an oil. This oil wasrehydrogenolysed in aqueous ethanol with 200 mg palladium on carbon for41/2 hours to afford a further 65 mg of the title compound; totalyield=330 mg (44%). (Nujol) 1805, 1705, 1695 (shoulder), 1615, 1580,1300, 1190, 1050, 1020, 1008, 895, 758 cm⁻¹, 'KBr) 1800, 1708, 1620,1590, 1300, 1203, 1190, 1125, 1050, 1020, 895 cm⁻¹. (D₂ O/DMSO D6,1.15-1.90 (6H, bm, NCH₂ (CH₂)₃ CH₂), 2.15-247 (2H, m, CH₂ CO₂ H),2.80-3.10 (2H, m, N.sup.⊕ CH₂ (CH₂)₃), 3.07 (1H, d, J 17 Hz, 6βCH),partially obscured), 3.58 (1H, dd, J 17 and 2.5 Hz, 6αCH, partiallyobscured), 3.67 (2H, d, J 8 Hz, 9CH₂), 8CH obscured by HOD at 4.60, 4.91(1H, s, 3CH), 5.74 (1H, d, J 2.5 Hz, 5αCH).

EXAMPLE 10 (a) Benzyl9-N-(3'-benzyloxycarbonylpropyl)-N-benzylaminodeoxyclavulanate

Benzyl dichloroacetylclavulanate (14.35 g; 36 mmol) in drydimethylformamide (100 cm³) at -10° was treated with 1.9 equivalents ofN,O-dibenzyl-4-aminobutyric acid (19.4 g dropwise in dimethylformamideover 10 minutes. Stirring was continued for 11/4 hours between -10° and0° C. The reaction mixture was poured into ethyl acetate (500 cm³) andwashed with water (4×100 cm³), saturated brine (6×150 cm³), dried(anhydrous magnesium sulphate) and evaporated in the presence of tolueneto low volume. This crude product was chromatographed on silica elutingwith toluene-ethyl acetate (4:1). Fractions were collected containingthe title compound Rf (SiO₂ /toluene-ethyl acetate; 4:1)=0.4 (detectionby aqueous potassium permanganate spray). Combined fractions wereevaporated to afford 5.6 g (28%) of an oil. (film) 1805, 1740, 1700,1500, 1455, 1307, 1172, 1015, 740, 700 cm⁻¹, (CDCl₃) 1.60-1.94 (2H, m,NCH₂ CH₂ CO₂), 2.15-2.50 (4H, m, NCH₂ CH₂ CH₂ CO₂), 2.91 (1H, d, J 17Hz, 6βCH), 3.12 (2H, d, J 7 Hz, 9CH₂), 3.35 (1H, dd, J 17 and 3 Hz,6αCH), 3.41 (2H, s, NCH₂ C₆ H₅), 4.68 (1H, bt, J 7 Hz, 8--CH), 5.03 (1H,s, 3CH), [5.05 (2H, s) and 5.14 (2H, s) 2×CO₂ CH₂ C₆ H₅ ], 5.57 (1H, d,J 3 Hz, 5αCH), [7.21 (5H, s) and 7.30 (10H, bs), 2×OCH₂ C₆ H₅ ].

(b) 9-N-(3'-Carboxypropyl)aminodeoxyclavulanic acid

Benzyl 9-N-(3'-benzyloxycarbonylpropyl)-N-benzylaminodeoxyclavulanate (2g; 3.6 mmol) in ethanol (30 cm³) tetrahydrofuran (15 cm³) and water (5cm³) was hydrogenolysed in the presence of 10% palladium on carbon (0.6g; which had been prehydrogenated for 10 minutes) for 21/2 hours atatmospheric pressure. The catalyst was filtered off and washed withethanol (30 cm³) then with water (50 cm³), this aqueous washing wascollected separately and was evaporated in the presence of ethanol to awhite crystalline solid. The solid was washed with cold ethanol anddried to afford 365 mg of the title compound. The initial filtrate andethanolic washings were rehydrogenated with the original catalyst for afurther 16 hours. The catalyst was filtered off and washed with aqueousethanol (20 cm³). The filtrate was evaporated to an oil, to whichmethanol was added and cooled (0°), crystals formed which were filteredoff, washed with cold methanol and dried to afford a further 103 mg ofthe required product. Total yield=0.47 g (46%), Rf (SiO₂ /ethylacetate-ethanol-water; 5:3:3)=0.33 (detection by aqueous potassiumpermanganate spray), (Nujol) 1802, 1900, 1595, 1575, 1300, 1187, 1122,1045, 1020, 1005, 915, 895, 852, 790, 760 cm⁻¹, (D₂ O) CH₃ CN internalstandard at 1.98; 1.70-2.10 (2H, m, NCH₂ CH₂ CH₂ CO₂ H), 2.31 (2H, t, J7 Hz, CH₂ CO₂ H), 3.00 (2H, t, J 7 Hz, N.sup.⊕ H₂ CH₂ CH₂), 3.07 (1H, d,J 17 Hz, 6βCH), 3.55 (1H, dd, J 17 and 3 Hz, 6αCH), 3.70 (2H, d, J 7 Hz,9CH₂), 4.78 (1H, bt, J 7 Hz, 8CH ), 4.99 (1H, s, 3CH), 5.73 (1H, d, J 3Hz, 5αCH).

EXAMPLE 11 (a) Benzyl9-N-(10'-benzyloxycarbonyldecyl)-N-benzylaminodeoxyclavulanate

Benzyldichloroacetylclavulanate (9.7 g; 24.2 mmol) in drydimethylformamide (80 cm³) at -10° was treated with 1.9 equivalents ofN,O-dibenzyl 11-aminoundecanoic acid (in 20 cm³ dimethylformamide)dropwise over 10 minutes and then at -10° to -5° over 40 minutes withvigorous stirring. The mixture was poured into ethylacetate (350 cm³)washed with water (5×150 cm³) and saturated brine (5×150 cm³), dried(anhydrous magnesium sulphate) and evaporated to an oil. This crudeproduct was chromatographed on silica eluting with ethylacetate-toluene(0:4), fractions were collected containing the title compound, Rf (SiO₂/ethylacetate-toluene; 1:4)=0.5 (detection by aqueous potassiumpermanganate spray). Combined fractions were evaporated to afford thetitle compound as an oil, yield=6.24 g (40%) ν (film) 1805, 1737, 1700(shoulder), 1305, 1170, 1005, 740, 700 cm⁻¹, δ(CDCl₃) 1.0-1.80 (16H,broad m, NCH₂ (CH₂)₈ CH₂ CO₂), 2.33 (4H, broad t, J 7 Hz, NCH₂ (CH₂)₈CH₂ CO₂), 2.93 (1H, d, J 17 Hz, 6βCH), 3.15 (2H, d, J 7 Hz, 9CH₂), 3.40(1H, dd, J 17 and 3 Hz, 6αCH), 3.43 (2H, s, NCH₂ C₆ H₅), 4.72 (1H, t, J7 Hz, 8CH), 5.05 (1H, s, 3CH), [5.08 (2H, s), 5.16 (2H, s) 2×OCH₂ C₆ H₅], 5.60 (1H, d, J 3 Hz, 5αCH), [7.24 (5H, s), 7.32 (10H, broad s), 2×OCH₂ C₆ H₅, NCH₂ C₆ H₅ ].

(b) 9-N-(10'-Carboxydecyl)aminodeoxyclavulanic acid

Benzyl 9-N-(10'-benzyloxycarbonyldecyl)-N-benzylaminodeoxyclavulanate (3g; 4.6 mmol) in ethanol (25 cm³), tetrahydrofuran (25 cm³) and water (5cm³) was hydrogenolysed at atmospheric pressure in the presence of 1 g10% palladium on carbon for 21/2 hours. The catalyst was filtered offand washed with ethanol; then with aqueous ethanol (150 cm³), thisaqueous washing was collected separately and evaporated to a whitecrystalline solid; this solid was washed with tetrahydrofuran-ethanoland dried to afford 430 mg (24%) of the title compound. The ehtanolicwashings and initial filtrate was rehydrogenated with the originalcatalyst for 30 minutes. The catalyst was filtered off and washed withaqueous ethanol, the filtrate was evaporated to an oil, tetrahydrofuran(10 cm³) was added and the solution cooled, crystals formed which werefiltered off cold and washed with tetrahydrofuran (0°), drying affordeda further 0.32 g (18%) of the title compound; total yield=0.75 g (42%)Rf (SiO₂ /ethylacetate-ethanol-water; 5:3:3)=0.70 (detection by aqueouspotassium permanganate spray), ν (Nujol) 1807, 1720, 1695, 1600 (broad),1298, 1182, 1060, 895, 750 cm⁻¹, ν (KBr) 1808, 1720, 1695, 1600, 1397,1295, 1183, 1130, 1057, 1022, 892, 750 cm⁻¹, δ(D₂ O/C₅ D₅ N; 2:1)0.75-1.90 (16H, broad m, NCH₂ (CH₂)₈ CH₂), 2.23 (2H, t, J 7 Hz, NCH₂(CH₂)₉ CH₂ CO₂ H), 2.99 (2H, t, J 7 Hz, NCH₂ (CH₂)₈ CH₂), 3.02 (1H, d, J17 Hz, 6βCH), 3.59 (1H, dd, J 17 and 3 Hz, 6αCH), 3.75 (2H, d, J 7 Hz,9CH₂), 8CH obscured by HOD at 4.95, 5.06 (1 H, s, 3CH), 5.85 (1H, d, J 3Hz, 5αCH); internal standard CH₃ CN at δ 2.00.

EXAMPLE 12 (a) O-Benzyl trans-4-(Aminomethyl)cyclohexane carboxylic acid

Trans-4-Aminomethyl cyclohexane carboxylic acid (25 g; 160 mmol) inbenzyl alcohol (100 cm³) and toluene (50 cm³) with para-toluenesulphonic acid monohydrate (30.2 g; 1 equivalent) was refluxed for 171/2hours using a Dean and Stark apparatus to collect the water ofcondensation and from the toluene sulphonic acid monohydrate. The clearreaction mixture was poured into ether (600 cm³) and the resultant whitesolid filtered off, washed with ether and dried to afford 57 g (85%) ofthe title compound. Both infrared and nuclear magnetic resonance spectrawere consistant with the desired compound.

(b) N,O-Dibenzyl-trans-4-aminomethylcyclohexanecarboxylic acid

O-Benzyl-4-trans aminomethyl-cyclohexanecarboxylic acid para-toluenesulphonate (30 g; 72 mmol) in CHCl₃ (150 cm³) was treated withtriethylamine (10.5 cm³ ; 1 equivalent), then with benzaldehyde (1equivalent), benzylalcohol (20 cm³) and stirred for 1 hour. To thereaction mixture was added excess sodium borohydride in small portions,the excess was destroyed by adding water until effervescence ceased. Thereaction mixture was diluted with chloroform (150 cm³) and washed withsaturated brine (5×150 cm³), dried (anhydrous magnesium sulphate) andevaporated to an oil. This oil was dissolved in diethyl ether (600 cm³)and treated with excess para-toluene sulphonic acid in ether. Theresultant white para-toluene sulphonate salt was filtered off, washedwith ether and dried to afford 27 g of a white crystalline solid. Thissolid was stirred vigorously in ethyl acetate-water whilst being treatedwith aqueous sodium hydroxide to pH 10. The ethyl acetate phase waswashed with brine (5×150 cm³) dried and evaporated to an oil whichcrystallised to an off white solid, yield=17 g (70%) (DBr) 3305, 1720,1450, 1256, 1185, 1125, 1010, 805, 747, 733, 695 cm⁻¹, δ(CDCl₃) 0.6-2.6(10H, m, C₆ H₁₀), 2.00 (1H, s, exchanges with D₂ O, NH), 2.45 (2H, d, J5 Hz, NCH₂ CH), 3.75 (2H, s, NCH₂ C₆ H₅), 5.09 (2H, s, OCH₂ C₆ H₅), 7.31(10H, bs, 2×CH₂ C₆ H₅). C₂₂ H₂₇ NO.sub. 2 requires 337.2040, 337.2035found, m/e 337 (M⁺), 120 (100% I), 106, 91, 65.

(c) Benzyl9-N-(4'-trans-benzyloxycarbonylcyclohexylmethyl)-N-benzylaminodeoxyclavulanate

Benzyldichloroacetylclavulanate (10.4 g; 26 mmol) in drydimethylformamide (80 cm³) at -10° was treated with 1.9 equivalents ofN,O-dibenzyl 4-trans aminomethylcyclohexanecarboxylic acid (16.6 g) indimethylformamide (50 cm³), dropwise over 10 minutes, then stirred for 3hours between -10° and +10°. The mixture was poured into ethyl acetate(300 cm³), dried (anhydrous magnesium sulphate) and evaporated to lowvolume in the presence of toluene. This crude product waschromatographed on silica eluting with toluene-ethylacetate (5:1),fractions were collected containing the title compound and combinedfractions were evaporated to afford the title compound as an oil; 3.94 g(25%), ν(film) 1805, 1735, 1700 (shoulder), 1455, 1305, 1175, 1010, 745,700 cm⁻¹. The proton magnetic resonance spectra was consistent with thedesired product.

(d) 9-N-(trans-4'-Carboxycyclohexylmethyl)aminodeoxyclavulanic acid

Benzyl 9-N-(trans-4'-benzyloxycarbonylcyclohexylmethyl)-N-benzylaminodeoxyclavulante (3.6 g; 5.9 mmol) in tetrahydrofuran (50 cm³) and water(5 cm³) was hydrogenolysed for 33 minutes at atmospheric pressure in thepresence of 10% palladium on charcoal (1.2 g; which had beenprehydrogenated for 10 minutes), the catalyst was filtered off andwashed with tetrahydrofuran (50 cm³), then separately with 200 cm³ of amixture of ethanol-tetrahydrofuran and water (1:1:2). This aqueouswashing was collected and evaporated to afford 1.0 g (50%) of the titlecompound as a white crystalline solid from cold ethanol. The initialreaction filtrate and tetrahydrofuran washings were evaporated to anoil, ethanol was added and on cooling crystals formed with when filteredoff and washed with cold ethanol afforded (after drying) a further 70 mgof the desired product. Rf (SiO₂ /ethylacetate:ethanol:water; 5:3:3)=0.4(detection by aqueous potassium permanganate spray), ν(Nujol) 1798,1695, 1610, 1580, 1305, 1187, 1127, 1055, 1020, 935, 100 cm⁻¹, ν(DBr)1795, 1700, 1600 (broad), 1452, 1400, 1302, 1185, 1125, 1020, 935, 895cm⁻¹. δ(D₂ O/pyridine d-5) 0.5-2.0 (10H, broad m, CH₂ C₆ H₁₀ CO₂ H),2.49 (2H, d, J 6 Hz, N.sup.⊕ CH₂ C₆ H₁₀), 2.70 (1H, d, J 17 Hz, 6βCH),3.23 (1H, dd, J 17 and 3 Hz, 6αCH), 3.37 (2H, d, J 8 Hz, 9CH₂), 8CHobscured by HOD at δ4.50, 4.67 (1H, s, 3CH), 5.46 (1H, d, J 3 Hz, 5αCH).

EXAMPLE 13 (a) Benzyloxycarbonylmethyl9-(N-benzyl-N-benzyloxycarbonylmethyl)amino-9-deoxyclavulanate

A stirred suspension of 9-amino-9-deoxyclavulanic acid (400 mg, 2.0mmol) in dry DMF (20 ml) was cooled to 0° and treated with DBN (750 mg,6.0 mmol). After 2 minutes the solution was treated with benzyl bromide(0.24 ml, 2.0 mmol) and stirred at 0° for 35 minutes before the additionof benzyl bromoacetate (1 ml). Stirring was continued for a further 21/2hours at room temperature.

The reaction mixture was diluted with ethyl acetate and washed well withwater. The ethyl acetate layer was dried (MgSO₄) and evaporated toafford an oil which was chromatographed on silica gel. Elution withEtOAc/cyclohexane (1:2) gave the desired product as an oil (109 mg, 9%).

ν_(max) (liq. film) 1800, 1750, 1700, 1160 cm⁻¹.

δ(CDCl₃) 2.87 (1H, d, J 17 Hz, β-lactam CHH), 3.30 (1H, dd, J 17 and 3Hz β-lactam CHH), 3.30 (2 h, s, NCH₂ Ph), 3.35 (2H, d, J 7 Hz, C(9)H),3.72 (2H, s, NCH₂ CO₂), 4.62 (2H, s, OCH₂ CO₂), 4.81 (1H, t, J 7 Hz,vinyl H), 5.09 (5H, s, OCH₂ Ph and C(3)H), 5.50 (1H, d, J 3 Hz, β-lactamCH), 7.29 (15H, s, aryl H) ppm.

Continued elution with the same solvent afforded the ester as an oil(168 mg, 13%). This material has been prepared previously.

(b) Lithium9-(N-benzyl-N-benzyloxycarbonylmethyl)amino-9-deoxyclavulante

A solution of the ester (i) above (100 mg) in aqueous tetrahydrofuranwas stirred at room temperature and treated with 1M LiOH solutiondispensed from an automatic burette at such a rate as to keep the pH ofthe solution to 10±0.5. The reaction was terminated when 1 equivalent ofLiOH had been consumed (3 hours). Dilute hydrochloric acid was thenadded dropwise to bring the pH to 7.0.

The resulting solution was evaporated to dryness and triturated withether to affored the desired salt as a white solid (62 mg, 80%).

ν_(max) (Nujol) 1790, 1730, 1710, 1610 cm⁻¹.

δ (D₂ O) 2.65 (1H, d, J 17 Hz, β-lactam CHH), 3.10 (2H, s, NCH₂ Ph), ca3.2 (3H, overlapping signals, β-lactam CHH and C(9)H), 3.52 (2H, s, NCH₂CO₂), 4.80 (1H, s, C(3)H), 4.87 (2H, s, OCH₂ Ph), 5.54 (1H, broad s,β-lactam CH), 7.13 and 7.18 (10H, aryl H) ppm.

(c) Lithium 9-N-(carboxymethyl)amino-9-deoxyclavulanate

A solution of lithium9-(N-benzyl-N-benzyloxycarbonylmethyl)amino-9-deoxyclavulanate (32 mg)in water (20 ml), containing THF (5 ml), was hydrogenated over 10%Pd/^(C) (20 mg) for 4 hours at ambient temperature and pressure. Thecatalyst was filtered off and the solution evaporated to afford thedesired product as a white solid (18 mg, 95%).

ν_(max) (KBr) 1780, 1720, 1620 cm⁻¹.

δ(D₂ O) 3.10 (1H, d, J 17 Hz, β-lactam CHH), 3.56 (1H, dd, J 17 and 3Hz, β-lactam CHH), 3.50 (2H, s, NCH₂ CO₂ H), 3.72 (2H, d, J 7 Hz,C(9)H), 4.79 (1H, t, J 7 Hz, vinyl H), 4.99 (1H, s, C(3)H), 5.73 (1H, d,J 3 Hz, β-lactam CH) ppm.

EXAMPLE 14 (a) Nα-Benzyloxycarbonyl-O-benzyl-(L)-lysine p-toluenesulphonate

Nα-Benzyloxycarbonyl (L) lysine (10 g; 35.7 mmol) in benzyl alcohol (30cm³), toluene (30 cm³) and para toluene sulphonic acid (6.93 g) washeated under reflux. The water evolved from the reaction and from thep-toluene sulphonic acid monohydrate was collected azeotropically usinga Dean and Stark apparatus. Refluxing was continued until the water wasno longer collected. The reaction mixture was allowed to cool, whencrystals formed. The crystals were filtered off and dried, yield=5 g.The proton magnetic resonance and infrared spectra showed this compoundto be the p-toluene sulphonic acid salt of the starting material. Thefiltrate was poured into ether (300 cm³) forming an oil. The ether wasdecanted off and the oil washed with ether and dried under reducedpressure. The resultant oil slowly crystallised to afford 12 g (62%) ofthe title compound, ν (Nujol) 3370, 1735, 1692, 1040, 1015, 820, 745,735, 700, 685 cm⁻¹.

The 5 g of recovered Nα-benzyloxycarbonyl para-toluene sulphonate wasre-enacted with benzyl alcohol (20 cm³), toluene (20 cm³) and 0.1equivalent of para-toluene sulphonic acid under reflux using a Dean andStark for 20 hours. The reaction mixture was cooled, then poured intoether, forming an oil, which crystallised slowly to afford a further 5.5g of the title compound. Total yield=17.5 g (90.4%).

(b) NΕ,O-dibenzyl-Nα-benzyloxycarbonyl(L) lysine ##STR10##

Nα-Benzyloxycarbonyl-O-benzyl(L) lysine para-toluene sulphonate (5.5 g)in water (100 cm³) and ethyl acetate (200 cm³) was treated with sodiumcarbonate to pH 10. The ethyl acetate phase was washed with saturatedbrine (3×200 cm³), dried (anhydrous magnesium sulphate) and evaporatedto an oil, yield=4.4 g of free amino compound. This was dissolved inchloroform (100 cm³), benzyl alcohol (10 cm³) and treated with 1.2equivalents of benzaldehyde, stirred for 2 hours then treated withexcess sodium borohydride. The solids were filtered off and the filtratewashed with saturated brine (5×150 cm³), dried and evaporated to an oil.This oil was dissolved in ether (300 cm³) and treated with para-toluenesulphonic acid in ether until in slight excess. An oil formed whichcrystallised at -10° overnight. The crystals were filtered off andwashed with dry ether, then treated with sodium carbonate whilststirring vigorously in ethyl acetate and water to pH 10. The organicphase was washed with saturated brine (4×100 cm³), dried and evaporatedto afford the title compound as an oil, yield=3.1 g (57%) ν(film) 3330(br), 1720, 750, 740, 700 cm⁻¹, δ(CDCl₃) 1.1-1.9 (6H, bm, NCH₂ (CH₂)₃CH), 1.20 (1H, s, exchanges+D₂ O; CH₂ NH), 2.52 (2H, bt, J 6 Hz, NCH₂(CH₂)₃), 3.69 (2H, s, NCH₂ C₆ H₅), 4.20-4.52 (1H, bm, ##STR11## 5.05 and5.11 (2×2H, 2×s, 2×CO₂ CH₂ C₆ H₅), 5.20-5.50 (1H, m, exchanges+D₂ O;NHCO₂), 7.25 (5H, s) and 7.28 (10H, s); 3×CH₂ C₆ H₅. C₂₈ H₃₂ N₂ O₄requires 460.2359; 460.2367 found m/e 460 (M+), 369 (M+-91), 308, 262,261, 174, 160, 155, 120, 106, 92, 91 (100% I), 65. The title compoundwas obtained as a white crystalline solid from ethylacetate-cyclohexane, ν (NUJOL) 3320 (broad), 1725, 1687, 1535, 750, 725,695 cm⁻¹. [α]_(D) ²⁰ c=1% in chloroform=+0.61°.

(c) Benzyl 9-[Nε,(Nε,O-dibenzyl-Nα-Z-(L)lysyl)]deoxyclavulanate

Benzyl dichloroacetylclavulanate (2.36 g; 5.9 mmol) in drydimethylformamide (30 cm³) at -10° was treated with 1.9 equivalents ofNε,O-dibenzyl-Nα-Z-(L) lysine, dropwise in dimethylformamide (40 cm³)over 10 minutes. Then stirred between -10° and +10° over 13/4 hours. Thereaction mixture was poured into ethylacetate (200 cm³) and washed withwater (5×100 cm³), saturated brine (3×100 cm³) dried (anhydrous)magnesium sulphate) and evaporated to an oil. This oil waschromatographed on silica eluting with toluene--ethyl acetate (3:1),fractions were collected containing the title compound Rf (SiO₂/toluene:ethyl acetate, 2:1)=0.45 (detection by aqueous potassiumpermanganate spray). Combined fractions were evaporated to afford anoil, 1.63 g (38%), ν (film) 3370 (br), 1800, 1745, 1722, 1515, 1495,1305, 1180, 1040, 1025, 1012, 740, 700 cm⁻¹, δ(CDCl₃) 1.0-2.4 (8H, bm,N(CH₂)₄ CH), 2.90 (1H, d, J 17 Hz, 6βCH), 3.10 (2H, d, J7 Hz, 9 CH₂),3.35 (1H, dd, J 17 and 3 Hz, 6αCH), 3.38 (2H, s, NCH₂ C₆ H₅), 4.17-4.50(1H, bm, CH₂ CH(NHCO₂ CH₂ C₆ H₅)), 4.68 (1H, t, J 7 Hz, 8CH), 5.02 (1H,s, 3CH), [ 5.07 (2H, s); 5.13 (4H, s), 2×CO₂ CH₂ C₆ H₅ and NCO₂ CH₂ C₆H₅ ], 5.20-5.40 (1H, bm, NH, partially obscured), 5.58 (1H, d, J 3 Hz,5αCH), [7.21 (5H, s); 7.29 (15H, s), 3×CO₂ CH₂ C₆ H₅ and NCH₂ C₆ H₅ ],[α]_(D) ²⁰ (c=1.2% in chloroform)=+5.4°.

(d) 9-[Nε(L)-Lysyl)]deoxyclavulanic acid

Benzyl 9-[Nε(Nε,O-dibenzyl-N-Z-(L)lysyl)]deoxyclavulanate (1.38 g; 1.89mmol) in ethanol (10 cm³), tetrahydrofuran (15 cm³) and water (3 cm³)was hdyrogenolysed in the presence of 10% palladised carbon (450 mmg;which had been prehydrogenolysed for 15 minutes) for 41/2 hours. Thecatalyst was filtered offf and washed with aqueous ethanol (100 cm³).The filtrate was evaporated to an oil which was redissolved in aqueousethanol (50 cm³) and rehydrogenolysed with 200 mg of fresh palladisedcarbon for 4 hours when thin layer chromatography showed the reaction tobe complete. The catalyst was filtered off and washed with water. Thefiltrate was evaporated to afford an oil, which on trituration withpropan-2-ol afforded the title compound as a cream coloured solid,yield=366 mg (59%) Rf (Sio₂ /ethylacetate--ethanol--water; 1:1:1)=0.33(detection by aqueous potassium permanganate spray) ν (KBr) 1790, 1690,1620 (very broad), 1400, 1310, 1192, 1120, 1044, 1017 cm⁻¹. The protonmagnetic resonance spectrum was consistent with the desired product.

EXAMPLE 15 2'-Phenylmethoxycarbonylmethyl-9-[N-(2'-phenylmethoxycarbonylmethyl)amino]-9-deoxyclavulanate(a) Methyl α-iodophenylacetate

Sodium iodide (7.5 g, 0.05 mol) dissolved in analar acetone (25 ml) wasstirred at room temperature and to it added methyl α-chlorophenylacetate(9.32 g, 0.05 mol). Stirred for 15 minutes. The precipitate sodiumchloride was allowed to settle.

(b) 2'-Phenylmethoxycarbonylmethyl-9-[N-(2'-phenylmethoxycarbonylmethyl)amino]-9-deoxyclavulanate

A suspension of 9-ADCA (198 mg, 1.0 mmol) in dry DMF (10 ml) was stirredat 0° and treated with DBN (375 mg, 3.0 mmol). After 2 minutes asolution of methyl α-iodophenylacetate in acetone (5 ml of abovesolution) was added in one portion and stirring at 0° maintained for 30minutes. The solution was then allowed to warm to room temperature andstirring continued for a further 21/2 hours.

The reaction mixture was diluted with ethyl acetate (ca 50 ml) andfiltered. The filtrate was evaporated to an oil which was againtriturated with ethyl acetate, filtered, and the solvent removed underreduced pressure. The resulting oil was chromatographed on silica geleluting with ethyl acetate/cyclohexane (1:2) to afford the desiredproduct as a yellow oil (22 mg, 5%).

ν_(max) (liquid film) 1800, 1740, 1690, 1220, 1170 cm⁻¹.

δ(CDCl₃) 1.90 (1H, broad s, NH), 3.02 (1H, d, J 17 Hz, β-lactam (CHH),3.32 (2H, d, J 7 Hz, C(9)H), 3.43 (1H, dd, J 17 and 3 Hz, β-lactam(CHH), 3.65 and 3.70 (6H, s, OCH₃), 4.31 and 4.42 (1H, s, NHCH), 4.95(1H, t, J 7 Hz, vinyl H), 5.13 (1H, complex, C(3)H), 5.64 (1H, d, J 7Hz, β-lactam CH), 5.93 and 5.99 (1H, s, --CO₂ CH), 7.30 and 7.39 (10H,s, Aryl H) ppm.

EXAMPLE 16 Di(ethoxycarbonyl)methyl9-N-[di(ethoxycarbonyl)methyl]amino-9-deoxyclavulanate

A suspension of 9-ADCA (198 mg, 1.0 mmol) in dry DMF (10 ml) was stirredat 0° and treated with DBN (250 mg, 2.0 mmol) in DMF (2 ml). After 2minutes diethyl bromomalonate (10 ml) was added and stirring at 0°maintained for 30 minutes. The reaction mixture was allowed to warm toroom temperature and stirring was continued for a further 21/2 hours.

The DMF was removed under reduced pressure and the residue dissolved inethyl acetate. After washing with a little water the ethyl acetate wasevaporated and the resultant oil chromatographed on silica gel elutingwith ethyl acetate/cyclohexane (1:3→1:1). The desired product wasobtained as an oil (11 mg, 2%).

ν_(max) (CHCl₃) 3300, 1805, 1750, 1740, 1690 cm⁻¹.

δ(CDCl₃) 1.23 (6H, t, J 7 Hz, CH₂ CH₃), 1.27 (6H, t, J 7 Hz, CH₂ CH₃),3.02 (1H, d, J 17 Hz, β-lactam CHH), 3.34 (2H, d, J 7 Hz, C(9)H), 3.45(1H, dd, J 17 and 3 Hz, β-lactam (CHH), 4.00 (1H, s, NHCH), 4.10-4.37(8H, complex, OCH₂ CH₃), 4.85 (1H, t, J 7 Hz, vinyl H), 5.15 (1H, s,C(3)H), 5.50 (1H, s, OCH), 5.66 (1H, d, J 3 Hz, β-lactam CH) ppm.

PHARMACOLOGY Synergistic Activity with Amoxycillin

Using conventional methods the following results were obtained in astandard MIC test:

    ______________________________________                                         Amoxycillin and                                                                           MIC μg/ml amoxycillin                                         Compound of  St aureus  K aerogenes                                                                              E coli                                     Example No   Russell    E70        JT39                                       ______________________________________                                        1 (b)  5.0 μg/ml                                                                            0.08       2.0      1.0                                             1.0 μg/ml                                                                            0.3        6.0      4.0                                      3 (c)  5.0 μg/ml                                                                            0.08       3.1      4.0                                             1.0 μg/ml                                                                            0.6        12.5     8.0                                      4 (c)  5.0 μg/ml                                                                            --         6.2      4.0                                             1.0 μg/ml                                                                            0.15       25       31.2                                     6 (b)  5.0 μg/ml                                                                            --         3.1      4.0                                             1.0 μg/ml                                                                            0.15       6.2      8.0                                      9 (b)  5.0 μg/ml                                                                            0.08       3.1      4.0                                             1.0 μg/ml                                                                            1.25       12.5     16                                       10 (b) 5.0 μg/ml                                                                            1.25       3.1      4.0                                             1.0 μg/ml                                                                            2.5        12.5     31.2                                     11 (b) 20 μg/ml                                                                             --         12.5     8.0                                             5.0 μg/ml                                                                            --         12.5     31.2                                     12 (d) 5.0 μg/ml                                                                            0.15       1.5      4.0                                             1.0 μg/ml                                                                            1.25       12.5     31.2                                     14 (d) 5.0 μg/ml                                                                            0.15       3.1      4.0                                             1.0 μg/ml                                                                            0.6        6.2      16                                       Amoxycillin alone                                                                          500        500        2000                                       ______________________________________                                    

The amines had no activity alone at the above concentrations.

Antibacterial Activity

Using conventional methods the following results were obtained in astandard MIC test:

    ______________________________________                                        Compound of                                                                              St aureus   K aerogenes                                                                              E coli                                      Example No Russell     E70        JT39                                        ______________________________________                                         1 (b)     31.0        >500       15                                           3 (c)     31.0        250        62.5                                         4 (c)     4.0         250        31.0                                         6 (b)     8.0         250        31.0                                         9 (b)     31.0        250        31.0                                        10 (b)     62.5        250        31.0                                        11 (b)     8.0         >500       >500                                        12 (d)     16          250        31.0                                        14 (d)     31.0        >500       125                                         ______________________________________                                    

We claim:
 1. A compound of formula (II): ##STR12## or a pharmaceuticallyacceptable salt or ester thereof wherein A is hydrogen or apharmaceutically acceptable salifyling or esterifying radical and X isalkylene of 1-12 carbon atoms optionally substituted by hydroxy, amino,or alkoxy of 1 to 6 carbon atoms, which substituents are not on thecarbon atom adjacent the nitrogen atom; or a C₅₋₇ cycloalkylene group.2. A compound according to claim 1 wherein X is an alkylene group of 1-7carbon atoms optionally substituted by a hydroxy or alkoxy of 1 to 6carbon atoms group which substituents are not on the carbon atomadjacent the nitrogen atom.
 3. A compound according to claim 2 wherein Xis an alkylene group of 1-7 carbon atoms.
 4. A compound according toclaim 3 wherein X is --CH₂ --CH₂ --.
 5. A compound according to claim 1wherein A is hydrogen or alkyl of 1 to 6 carbon atoms.
 6. A compoundaccording to claim 5 wherein A is hydrogen.
 7. Lithium9-N-(carboxymethyl)amino-9-deoxyclavulanate.
 8. The compound accordingto claim 1 which is:9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid,9-N-(4'-Carboxybutyl)aminodeoxyclavulanic acid,9-N-(2'-Methoxycarbonylethyl)aminodeoxyclavulanic acid, Methyl9-N-(2'-carboxyethyl)aminodeoxyclavulanate,9-N-(5'-Carboxypentyl)aminodeoxyclavulanic acid,9-N-(3'-Carboxypropyl)aminodeoxyclavulanic acid,9-N-(10'-Carboxydecyl)aminodeoxyclavulanic acid, or 9-[Nε(L)-Lysyl]deoxyclavulanic acid.
 9. A pharmaceutical composition usefulfor treating bacterial infections in mammals including humans and foreffecting beta-lactamase inhibitory activity in mammals including humanswhich comprises a therapeutically effective amount of a compound of theformula (II): ##STR13## a pharmaceutically acceptable salt thereof or apharmaceutically acceptable ester thereof, wherein A is hydrogen or apharmaceutically acceptable salifyling or esterifying radical and X isalkylene of 1-12 carbon atoms optionally substituted by hydroxy, amino,or alkoxy of 1 to 6 carbon atoms, which substituents are not on thecarbon atom adjacent the nitrogen atom; or a C₅₋₇ cycloalkylene group,in combination with a pharmaceutically acceptable carrier.
 10. Acomposition according to claim 9 wherein X is an alkylene group of 1-7carbon atoms optionally substituted by a hydroxy or alkoxy of 1 to 6carbon atoms group which substituents are not on the carbon atomadjacent the nitrogen atom.
 11. A composition according to claim 9wherein X is an alkylene group of 1-7 carbon atoms.
 12. A compositionaccording to claim 9 wherein X is --CH₂ --CH₂ --.
 13. A compositionaccording to claim 9 wherein A is hydrogen or alkyl of 1-6 carbon atoms.14. A composition according to claim 9 wherein A is hydrogen.
 15. Acomposition according to claim 9 wherein the compoundis:9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid,9-N-(4'-Carboxybutyl)aminodeoxyclavulanic acid,9-N-(2'-Methoxycarbonylethyl)aminodeoxyclavulanic acid, Methyl9-N-(2'-carboxyethyl)aminodeoxyclavulanate,9-N-(5'-Carboxypentyl)aminodeoxyclavulanic acid,9-N-(3'-Carboxypropyl)aminodeoxyclavulanic acid,9-N-(10'-Carboxydecyl)aminodeoxyclavulanic acid, or9-[Nε(L)-Lysyl]deoxyclavulanic acid.
 16. A pharmaceutical compositionuseful for treating bacterial infections in mammals including humans andfor effecting beta-lactamase inhibitory activity in mammals includinghumans which comprises a therapeutically effective amount of9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid.
 17. A method of treatingbacterial infections in mammals including humans which comprisesadministering to such a mammal in need thereof an antibacteriallyeffective amount or a beta-lactamase inhibitory amount of a compound ofthe formula (II): ##STR14## a pharmaceutically acceptable salt thereofor a pharmaceutically acceptable ester thereof, wherein A is hydrogen ora pharmaceutically acceptable salifyling or esterifying radical and X isalkylene of 1-12 carbon atoms optionally substituted by hydroxy, amino,or alkoxy of 1 to 6 carbon atoms, which substituents are not on thecarbon atom adjacent the nitrogen atom; or a C₅₋₇ cycloalkylene group,in combination with a pharmaceutically acceptable carrier.
 18. A methodaccording to claim 17 wherein X is an alkylene group of 1-7 carbon atomsoptionally substituted by a hydroxy or alkoxy of 1 to 6 carbon atomsgroup which substituents are not on the carbon atom adjacent thenitrogen atom.
 19. A method according to claim 17 wherein X is analkylene group of 1-7 carbon atoms.
 20. A method according to claim 17wherein X is --CH₂ --CH₂ --.
 21. A method according to claim 17 whereinA is hydrogen or alkyl of 1-6 carbon atoms.
 22. A method according toclaim 17 wherein A is hydrogen.
 23. A method according to claim 17wherein the compound is:9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid,9-N-(4'-Carboxybutyl)aminodeoxyclavulanic acid,9-N-(2'-Methoxycarbonylethyl)aminodeoxyclavulanic acid, Methyl9-N-(2'-carboxyethyl)aminodeoxyclavulanate,9-N-(5'-Carboxypentyl)aminodeoxyclavulanic acid,9-N-(3'-Carboxypropyl)aminodeoxyclavulanic acid,9-N-(10'-Carboxydecyl)aminodeoxyclavulanic acid, or9-[Nε(L)-Lysyl]deoxyclavulanic acid.
 24. A method of treating bacterialinfections in mammals including humans and for effecting beta-lactamaseinhibitory activity in mammals including humans which comprisesadministering to a mammal in need thereof a therapeutically effectiveamount of 9-N-(2'-Carboxyethyl)aminodeoxyclavulanic acid.